Angiotensin II antagonists used to treat reproductive functions associated with AT2 receptors

ABSTRACT

The present invention relates to the use of 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acids and analogs thereof in antagonizing the binding of angiotensin II to AT2 receptors.

This application is a continuation-in-part of copending application Ser.No. 07/760,585 filed Sep. 19, 1991, now abandoned, which is acontinuation-in-part of application Ser. No. 07/591,928 filed Oct. 2,1990, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel utilities for derivatives of4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid thathave previously been disclosed to be useful for the treatment ofhypertension in U.S. Pat. No. 4,812,462 to Blankley et al, issued Mar.14, 1989. These compounds have the property of antagonizing the bindingof angiotensin II (angiotensin II), a peptide hormone, to one subtype ofits cellular receptors. In particular, the utility of these compoundsarises from activity at the AT₂ receptor as described by Bumpus, et al,in Hypertension, 1991, 17, 720-721.

The present invention is related to the discovery that the AT₂ receptoris found in the central nervous system (CNS) of mammals, and thatcompounds of general Formula I described herein are effective inblocking angiotensin II binding at AT₂ receptors in various brainregions. The present invention is also related to the discovery thatalterations in brain biochemistry are observed upon administration ofcompounds of general Formula I and that these alterations coincide withphysiological and behavioral responses.

The present invention is also related to the discovery that AT₂receptors are found in female reproductive organs of meals includinguterus (Data in Table 1, hereof and in Dudley, et al, MolecularPharmacol., 1990, 3.8, 370-377) and ovaries (Pucell, et al,Endocrinology, 1991, 128, 1947-59). The role of angiotensin II inprocesses leading to ovulation has been reviewed by Andrade-Gordon, etal, in Biochemical Pharmacology, 1991, 42, 715-719. Compounds of generalFormula I, inhibit the binding of angiotensin II to AT₂ receptors inreproductive tissues, including uterus and ovarian follicles and henceantagonize the effects of angiotensin II therein.

Finally, the present invention is related to the discovery that the AT₂receptor is found in neuronal tumor cells (Speth, et al, PeptideResearch, 1989, 2, 232-239) and in transformed human neural cells(Tallant, et al, Hypertension, 1991, 17, 1135-1143).

SUMMARY OF THE INVENTION

The present invention relates to the use of the compounds of generalFormula I which are set forth in Chart I hereof. The present inventionembraces the use of pharmaceutically acceptable salts of the compoundsof Formula I also.

The compounds of Formula I and pharmaceutically acceptable salts thereofhave been found to antagonize the binding of angiotensin II to AT₂receptors. Such a finding renders the compounds useful in treatingconditions which are associated with or result from the binding ofangiotensin II to AT₂ receptors. Thus, compounds of Formula I andpharmaceutically acceptable salts thereof are useful in treatingdisorders of the CNS which are attributed to the binding of angiotensinII to brain AT₂ receptors. The compounds of general Formula I andpharmaceutically acceptable salts thereof are additionally useful intreating conditions of the female reproductive system which result fromthe binding of angiotensin II to AT₂ receptors in reproductive organs.The compounds of general Formula I, radioactive isotopes thereof, andpharmaceutically acceptable salts thereof are also useful as tumorimaging and anticancer agents for brain cancers and other cancerswherein the AT₂ receptor is prevalent.

DETAILED DESCRIPTION OF THE INVENTION

Angiotensin II is known to modulate CNS nerve sensitivity toneurotransmitters such as catecholamines, serotonin and enkephalins, andadditionally, angiotensin II is a neurotransmitter that regulates therelease of hormones from the brain (Phillips, Ann. Rev. Physiol. , 1987,49, 413-35 and Septh et al, Research Topics in Physiology, 1988, 10,1-34). Agents that block the activity of angiotensin II at AT₂ receptorsin the CNS will ameliorate disorders associated with abnormal nerveactivity and abnormal hormone secretion related to exaggerated AT₂mediated responses to angiotensin II. The compounds of general FormulaI, being AT₂ antagonists have utility in the treatment and diagnosis ofnumerous neurological, psychiatric, neuroendocrine, neurodegenerativeand neuroimmunological disorders including, but not limited to, thoseassociated with addiction, anxiety, depression, epilepsy, hyperactivity,memory, pain, Parkinsonism, psychosis, regulation of autonomicfunctions, sleep and tardive dyskinesia.

Barnes, et al, in Brain Research, 1990, 507, 341-343, describe theeffects of angiotensin II as an inhibitor of potassium stimulatedrelease of ACh from human temporal cortex, giving rise to elevatedlevels of ACh in cortical tissue. Data from Tables IV, V, and VI hereofshow the dose related effect of PD 123319 (Example 1), a compound ofgeneral Formula I, in lowering ACh levels in rat striatum andhippocampus. This effect is similar to that described by Usinger, et al,in Drug Dev. Res., 1988, 14, 315-324, wherein rats treated with anangiotensin converting enzyme (ACE) inhibitor, a drug that blocks theformation of angiotensin II, also show reductions in striatal ACh.Usinger relates the ACh reduction in brain tissues to the memoryenhancing effects of ACE inhibitors. ACE inhibitors have been shown toenhance cognitive performance in rodent tests of cognition by Costall,et al, in Pharmacol. Biochem. & Behavior, 1989, 33, 573-579.

Since both ACE inhibitors and angiotensin receptor antagonists block theACh accumulating action of angiotensin II in the brain, it is reasonablethat both will enhance cognitive performance. Barnes, et al, inNeuroreport, 1991, 2, 351-353, have demonstrated that PD 123177 (Example2), a compound of general Formula I, does indeed possess cognitionenhancing actions in rodent behavioral models and that this compoundalso reverses scopalamine-induced amnesia.

Data from Table IV hereof shows that PD 123319 (Example 1), a compoundof general Formula I, matches the ACh lowering ability of haloperidol, adrug used in the treatment of psychotic disorders. It is thereforereasonable that compounds of general Formula I will have utility in thetreatment of psychotic disorders.

Another known CNS effect of angiotensin II is stimulation of the releaseof pituitary and hypothalamic hormones including vasopressin (AVP),oxytocin, adrenocorticotrophic hormone (ACTH), prolactin and luteinizinghormone (LH) (Speth, et al, Research Topics in Physiology, 1988, 10,1-34). Thus, compounds of general Formula I have utility in treatment ofvarious neuroendocrine disorders that are dependant upon the release ofhormones resulting from angiotensin II stimulation of AT₂ receptors.

Vasopressin (AVP), also known as antidiuretic hormone, is a peptidehormone which causes decreased urinary output, increased urine densityand reduced thirst. In normal physiology, it is important forconservation of body fluid (Laszlo, et al, Pharmacological Rev., 1991,43, 73-108). Schiavone, et al, in Hypertension, 1991, 17, 425, describethe effects of AT₂ antagonists including PD 123177 (Example 2), acompound of general Formula I, in antagonizing the angiotensin IIinduced secretion of AVP from isolated rat hypothalamo-neurohypophysialexplants. Data in Table VII, hereof, shows the increase in renal freewater clearance following administration of Example 1, as aphysiological correlate to the blockade of angiotensin II induced AVPsecretion. Excessive secretion of AVP has been linked to a number ofdisorders including excessive water retention associated with the femalereproductive disorder known as premenstrual syndrome (PMS) (Janowski, etal, Psychosomatic Medicine, 1973, 35, 143-154) and impaired waterexcretion with adrenal insufficiency (Schrier and Bichet, J. Lab. Clin.Med., 1981, 98, 1-15). It has also been linked to Schwartz-Barttersyndrome (an AVP secreting brain tumor), congestive heart failure, livercirrhosis, nephrotic syndromes, central nervous injuries, acutepsychotic states, lung disease, dysmenorrheic uterine hyperactivity, andpremature labor (Lazlo, et al, ibid.). Compounds of general Formula I,by virtue of their ability to block angiotensin II induced AVPsecretion, have utility in treatment of the above disorders.

AT₂ receptors are known to exist in organs of the female reproductivesystem. Data shown in Table I, hereof, provides evidence of angiotensinII receptor binding inhibition by compounds of general Formula I inrabbit uterine preparations. According to Dudley, et al, in MolecularPharmacology, 1990, 38, 370-377) this binding inhibition is specific forAT₂ (DTT insensitive) receptors. Pucell, et al, in Endocrinology, 1991,128, 1947-59) show AT2 antagonistic properties of PD 123319 (Example 1),a compound of general Formula I, in ovarian cells. More specifically,AT₂ receptors are densely located on granulosa cells in atreticfollicles of the ovary.

The developmental anatomy and physiology of ovarian follicles isdiscussed in Guyton's Textbook of Medical Physiology, 6th ed., pp1005-1020 (1981). Follicular granulosa cells are the progenitor of thecorpus lutes. They are known to differentiate into the progesteronesecreting cells of the corpus luteum upon ovulation. Data provided inTable IX, hereof, show a divergence of AT₁ and AT₂ receptor populationsbetween corpus lutea (AT₁ rich) and granulosa cells from atreticfollicles (AT₂ rich). Thus, angiotensin receptor subtypes are involvedin the regulation of reproductive processes in the ovary includingfollicle maturation, corpus luteal differentiation, hormonal cycles andfollicle atresia.

Table X, hereof, describes the results of autoradiography studies thatshow the ability of Example 12 to penetrate the ovary and bindspecifically to follicles. Compounds of general Formula I, by virtue oftheir AT₂ antagonistic properties and their ability to penetrate ovariantissues, have utilities associated with the reproductive functions ofovaries including, but not limited to, menstruation, fertility, anddisturbances of normal hormonal balances of the estrus cycle.

Table XI, hereof, describes the results of reproductive studies in ratswhere a representative compound of the present invention, particularlythe compound of Example 1, interrupts the menstrual cycle indicating itseffectiveness in relating fertility.

In contrast to the AT₁ receptor, which is coupled to G proteins andassociated with inositol trisphosphate (IP₃) metabolism, the AT₂receptor has been shown to lack G protein coupling and is dissociatedfrom IP₃ metabolism (Dudley, et al, Molecular Pharmacology, 1990, 38,370-377). IP₃ -dissociated angiotensin II receptors have been reportedto exist in neuronal tumor cells by Speth, et al, in Peptide Research,1989, 2, 232-239. Additionally, Tallant, et al, in Hypertension, 1991,17, 1135-1143, describe a dramatic upregulation of AT₂ receptors whenNG108-15 cells, a neurally derived clonal cell line, are induced todifferentiate by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate.

Given the numerous effects associated with CNS AT₂ receptors alreadydescribed above, excessive expression of these receptors in neuronalcancers would lead to pathological states. Compounds of general FormulaI by virtue of their AT₂ antagonizing properties have utility indisrupting the function of tumor cells that contain AT₂ receptors.Therefore, they are useful in blocking the actions and/or growth ofneuronal tumors as well as other tumors wherein the AT₂ receptor isprevalent.

Additionally, radioisotopically labeled compounds of general Formula Ihave utility as tumor imaging agents and as drugs for tumor-selectiveirradiation therapy. In both of these utilities, a radioactive atom isattached to a compound of general Formula I which has high affinity forAT₂ receptors on the tumor cells. Strategies for tumor imaging agentsinclude the incorporation of ¹²⁵ I ⁷⁷ Br and ¹⁸ F radiolabels asdescribed in FIG. 1. A compound of general Formula I containing one ofthese radiolabels carries nonlethal radiation to the AT₂ containingtumor where it enables detection of the cancerous site. The synthesis of¹²⁵ I-Example 12 is described below. Drugs for tumor-selectiveirradiation therapy involve incorporation of radioactive isotopes thatemit sufficient radioactivity to kill cells such as ¹³¹ I as describedin FIG. 2. A compound of general Formula I bearing ¹³¹ I specificallybinds to tumors bearing AT₂ receptors, resulting in irradiation of thetumor and regression of the cancerous lesion. The synthesis of ¹³¹I-Example 12 is analogous to the synthesis of ¹²⁵ I-Example 12 describedbelow. Radioisotopically labeled compounds of general Formula I, thushave utility as agents for imaging tumors or as agents that selectivelykill tumor cells by irradiation in cancerous states wherein AT₂receptors are prevalent.

The compounds of general Formula I are described and claimed in U.S.Pat. No. 4,812,462 which issued Mar. 14, 1989, which patent isincorporated herein by reference thereto. The compounds of generalFormula I and pharmaceutically acceptable salts thereof are described inU.S. 4,812,462 beginning at column 1, line 56 through column 4, line 48.The DETAILED DESCRIPTION OF INVENTION Section of U.S. Pat. No. 4,812,462describes method of synthesis of the compounds of Formula I (column 10,line 5 through column 17, line 27) and the manner of formulating thecompounds, the route of administration and the dosage amount. Inpracticing the present invention one would use the same dosage amounts,routes of administration and types of pharmaceutical composition as aredescribed in U.S. 4,812,462 (see column 20, line 7 through column 22,line 27 ).

Receptor Binding Data

The ability of the incident compounds to displace ¹²⁵ I- or ³ H-labeledangiotensin II from cell membrane preparations derived from rabbituterus and corpus 35 luteum was measured according to the methodsdescribed by Dudley, et al, (Molecular Pharmacology, 1990, 38, 370-377).Table I shows examples of IC₅₀ values for displacement of ³H-angiotensin II from rabbit uterus receptor preparations and Table IXcompares the relative populations of AT₁ and AT₂ in ovarian granulosacells and corpus luteum. Granulosa cells are rich in AT₂ receptors asdemonstrated by the fact that Example 1 (an AT₂ specific agent)displaces nearly all of the ¹²⁵ I-angiotensin II. Conversely, corpusluteum is rich in AT₁ receptors as demonstrated by the fact that DUP 753(an AT₁ specific agent) displaces nearly all of the radioligand.

Using the same receptor binding methodology, Table VIII compares theability of Example 1 (an AT₂ specific agent), DUP 753 (an AT₁ specificagent) and saralasin (an agent lacking receptor subtype specificity) todisplace ¹²⁵ I-labeled angiotensin II from receptor preparations derivedfrom various segments of rat brain. The concentration of all threeantagonist compounds in these experiments is 1 μM (above the maximaleffect concentration). Both Example 1 and Dup 753 displace a fraction ofthe ¹²⁵¹ -angiotensin II that is displaced by saralasin (Sat),indicating the presence of both receptor subtypes in each area of brainexamined.

Table II shows examples or IC₅₀ values for the incident compounds inwhole rat brain. Since receptor-specific binding in whole brain is lowand is a mixture of two subtypes, an alternative protocol (Bennett andSnyder, Eur. J. Pharmacol., 67, 11-25) was used. This method includesDTT and thereby eliminates binding at AT₁ receptors, thus allowingevaluation of AT₂ receptor events. The whole brain assay suffers from alow specific to nonspecific binding ratio relative to assays run inother tissues but it is possible to demonstrate the inhibitory action ofcompounds of general Formula I on the binding of angiotensin II to AT₂receptors. In summary, the above receptor binding data show that thesecompounds interact with AT₂ receptors in both brain and reproductivetissues.

Autoradiography

¹²⁵ I-Labeled Example 12 was prepared from Example 13 according to thefollowing procedure: A solution of Example 13 (2.0 μg, 4.1 nMol) inmethanol (2 μL), was treated with a solution of Na¹²⁵ I (5.0 mCi, 2.3nMol ) in potassium phosphate buffer (0.5 M, pH 7.2, 40 μL). Theiodination was initiated by addition of chloramine-T (30 μg, 132 nMol)that was diluted to a concentration of 0.05 M in the same buffer asabove. After shaking at room temperature for one minute the reaction wasquenched with sodium metabisulfite (10 mg). HPLC purification of thereaction mixture on an Altech RPC C₁₈ reversed phase column with anisocratic mobile phase of acetonitrile (30%) and 0.1% aqueoustrifluoroacetic acid (70%) afforded pure ¹²⁵ I-Example 12 as itstrifluoroacetate salt upon evaporation of solvents. This labeledmaterial was reconstituted in 5% aqueous dextrose containing 30 mg/mL ofthe disodium salt of unlabeled Example 12 and adjusted to pH 9 byaddition of several μL of 1 mM NaOH.

Male and female Sprague-Dawley rats (Charles River, Wilmington, averageweight 281 g) were used in the autoradiography studies. Animals were ona fixed light cycle with unrestricted access to normal rat chow andwater prior to the experiment. Rats received 30 mg/kg ¹²⁵ I-labeledExample 12 (966 μCi/kg), injected via the tail vein over one minute.Animals were killed at 15 or 60 minutes postdose by halothane anesthesiaand were rapidly frozen in acetone with dry ice. Carcasses weresubsequently embedded in methylcellulose ice for whole-body sectioningand autoradiography. Autoradiograms were analyzed by computer assisteddensitometry.

Results of the autoradiographical experiments are summarized in Table X.A relative scale of radioactivity is assigned with background equal to 0and the most dense reading (liver and intestines) equal to 10. The sitesthat are highly labeled by ¹²⁵ I-Example 12 include liver, intestinesand bladder which are all indicative of excretory processes since thesetissues show negligible AT₂ receptors in binding experiments. Anothersite that is highly labeled is the ovarian follicle. It is the mosthighly labeled non-excretory organ. All other tissues show weakerradioactivity. These results demonstrate the ability of Example 12 toselectively distribute to the ovarian follicle, a female reproductiveorgan.

Effects of Example 1 on Estrous Cycles in the Mature Rat

Female rats underwent vaginal smearing every morning for 5 weeks; ratswith regular 4-day cycles (proestrus, estrus, metestrus, diestrus) werechosen for further study. Groups of rats (n=5) were implanted with Alzetosmotic minipumps (Model 2ML2) containing Example 1 or vehicle on theafternoon of estrus (approximately 2 PM). Drug-treated animals received30 mg/kg/day Example 1 for 14 days. Cycles were continually monitoredthroughout the treatment period and for an additional 20 to 22 daysposttreatment.

Data are presented in Table XI. Vehicle-treated animals continued tocycle in a 4-day pattern throughout the study. Example 1-treated ratswere acyclic during the 14-day treatment interval but resumed cyclingduring the posttreatment observation period.

Effects of Example 1 on Brain ACh Levels

Example 1 was given by IP administration to normal rats at 1,10, 30 and60 mg/kg. Thirty minutes after dosing, the rats were sacrificed byexposure to microwave irradiation. ACh levels in striatum andhippocampus were then measured by HPLC assay with electrochemicaldetection according to the method of Beley, et al, (J. Liquid Chrom.,1987, 10, 2977) using the ACh analysis system from BioanalyticalSystems, Inc. Table IV shows the dose responsive nature of ACh reductionfollowing treatment with Example 1. Haloperidol (2 mg/kg, IP) provides apositive control. DUP 753 (an AT₁ specific antagonist of angiotensin II)shows no effect at 30 mg/kg, IP. Example 1 matched the efficacy ofhaloperidol, a known antipsychotic drug, in lowering striatal ACh. TableV shows a comparable lowering of ACh levels in the hippocampus andstriatum by Example 1 at a dose of 30 mg/kg. Table VI shows the timecourse of the ACh lowering action of Example 1. Peak effect is at 0.5hours, with ACh levels gradually returning to control levels in 24hours.

The data in Tables IV, V and VI indicate that Example I, a compound ofgeneral Formula I, inhibits the normal role of angiotensin II in braintissues. Normally, angiotensin II inhibits release of ACh from braintissues thereby elevating ACh levels in brain tissues. Example I, an AT₂specific agent lowers ACh content in brain tissues in a dose dependantand time dependant manner. These data provide evidence in support of theuse of compound of general Formula I in treatment of conditions whereinthe CNS responsiveness to angiotensin II is abnormal and leads toinappropriate elevation of brain ACh content, including, but not limitedto, the treatment of memory disorders and psychoses.

Effects of Example 1 on Renal Free Water Clearance

Dogs were anesthetized with sodium pentobarbital after an overnightfast. Animals were placed on a prewarmed surgical table, intubated andprepared for renal clearance experiments. Femoral arteries werecannulated for aortic blood pressure measurements and for bloodsampling. A femoral vein was cannulated and used for the infusion ofinulin. A left flank. incision was made to expose the kidney and ureter.An electromagnetic flow probe was placed on the left renal artery andthe left ureter was cannulated for urine collection. An intrarenalinfusion of dextrose in water (D5W, 50 μL/min) was administeredthroughout the protocol except during drug treatment intervals. Apriming dose of 50 mL D5W containing 200 mg/kg inulin was administeredto each dog via the femoral vein catheter and then followed by asustained infusion of inulin in DSW (15 mg/mL, 0.075 mL/min/kg).

Following a 45 minute equilibration period, a series of ten 15-minuterenal clearance periods were obtained. A clearance consisted of a timedurine collection with a midpoint blood sample. The first two 15-minuteclearances, during which vehicle was infused, were designated aspredose. Separated groups of animals received vehicle (control) orExample 1 in three cumulative doses (3, 30 and 300 μg/kg/min, n=5). Eachdose was infused intrarenally over 35 minutes, the animals were allowedto stabilize for 5 minutes, and two 15-minute clearances were collected.After the final dose, D5W was infused for a 60 minute washout and duringtwo 15-minute clearances. These last two clearances were designated aspostdose.

Plasma and urinary electrolytes were determined with an ion selectiveelectrode analyzer. Urine and plasma inulin concentrations were assayedusing an anthrone colorimetric method (Davidson, et al, J. Lab and Clin,Med., 1963, 62, 351-356). Renal clearances and fractional excretion werecalculated using standard formulas (Vander, Renal Physiology., 4th ed,(pp 44-52 (1991)). Table VII shows the free water clearance data foranimals of the vehicle control group and of the Example 1 treated group.The vehicle control animals show a slight but statisticallyinsignificant reduction in free water clearance as the experimentprogressed. The Example 1 treated animals show an increase in free waterclearance that is dose dependant and statistically significant whencompared to the vehicle control. Free water clearance is increased by upto 370% from predose levels upon treatment with Example 1. These dataprovide evidence in support of the use as a treatment for conditionswherein excessive responsiveness to angiotensin II at AT₂ receptorsresults in inappropriate water retention, including, but not limited to,neurohormonal conditions related to excessive vasopressin secretion suchas premenstrual syndrome, impaired water excretion with adrenalinsufficiency and Schwartz-Bartter syndrome.

                  TABLE I                                                         ______________________________________                                        IC.sub.50 Values in Rabbit Uterine Preparations                                ##STR1##                                                                     Ex   R.sub.1          R.sub.5      IC.sub.50 (M)                              ______________________________________                                        1    3-Me-4-NMe.sub.2PhCH.sub.2                                                                     COCHPh.sub.2 2.1 × 10.sup.-8                      2    3-Me=4-NH.sub.2PhCH.sub.2                                                                      COCHPh.sub.2 3.2 × 10.sup.-8                      3    3-Me-4-MeOPhCH.sub.2                                                                           COCHPh.sub.2 2.6 × 10.sup.-8                      4    1-Adamantylethyl COCHPh.sub.2 9.1 × 10.sup.-8                      5    3-Me-4-NMe.sub.2PhCH.sub.2                                                                     COCH(4-FPh).sub.2                                                                          3.8 × 10.sup.-8                      6    3-Me-4-MeOPhCH.sub.2                                                                           CO(9-Fluorenyl)                                                                            1.2 × 10.sup.-6                      7    3-Me-4-NO.sub.2PhCH.sub.2                                                                      COCHPh.sub.2 6.7 × 10.sup.-8                      8    PhCH.sub.2       COCHPh.sub.2 7.4 × 10.sup.-8                      9    3-Me-4-MeOPhCH.sub.2                                                                           COCH.sub.2 Ph                                                                              9.5 × 10.sup.-8                      10   3-Me-4-MeOPhCH.sub.2                                                                           COCH(4-ClPh).sub.2                                                                         1.2 × 10.sup.-7                      11   3-Me-4-MeCONHPhCH.sub.2                                                                        COCHPh.sub. 2                                                                              1.5 × 10.sup.-7                      12   3-I-4-HO-5-MePhCH.sub.2                                                                        COCHPh.sub.2 2.5 × 10.sup.-8                      ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        IC.sub.50 Values in Whole Rat Brain Preparations                               ##STR2##                                                                     Example    R.sub.1         IC.sub.50 (M)                                      ______________________________________                                         1         3-Me-4-Me.sub.2 NPhCH.sub.2                                                                   2.0 × 10.sup.-7                               4         1-Adamantylethyl                                                                              1.8 × 10.sup.-7                              12         3-Me-4-NH.sub.2PhCH.sub.2                                                                     5.7 × 10.sup.-7                              15         4-CF.sub.3PhCH.sub.2                                                                          7.8 × 10.sup.-7                              16         3-MePhCH.sub.2  1.6 × 10.sup.-6                               8         PhCH.sub.2      3.8 × 10.sup.-6                              ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Compound Names                                                                Example   Name                                                                ______________________________________                                         1        (S)-(-)-1-[[4-dimethylamino)-                                                 3-methylphenyl]methyl]-5-(diphenylacetyl)-                                    4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-                                         pyridine-6-carboxylic acid                                           2        (S)-(-)-1-[(4-amino-3-methylphenyl)-                                          methyl]-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-lH-imidazo[4,5-c]pyridine-6-                                       carboxylic acid                                                      3        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-[(4-methoxy-3-methyl-                                            phenyl)methyl]-1H-imidazo[4,5-c]pyridine-                                     6-carboxylic acid                                                    4        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-(2-tricyclo[3.3.1.1.sup.3,7 ]dec-1-                              ylethyl-1H-imidazo[4,5-c]pyridine-6-                                          carboxylic acid                                                      5        (S)-(-)-5-[bis(4-fluorophenyl)acetyl]-1-                                      [[4-(dimethylamino)-3-methyl-                                                 phenyl]methyl]-4,5,6,7-tetrahydro-1H-                                         imidazo[4,5-c]pyridine-6-carboxylic acid                             6        (S)-(-)-5-(9H)-fluoren-9-ylcarbonyl)-                                         4,5,6,7-tetrahydro-1-[(4-methoxy-3-                                           methylphenyl)methyl]-1H-imidazo-                                              [4,5-c]pyridine-6-carboxylic acid                                    7        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-[(3-methyl-4-nitrophenyl)-                                       methyl]-4-nitrophenyl)methyl]-1H-                                             imidazo[4,5-c]pyridine-6-carboxylic acid                             8        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-(phenylmethyl)-1H-                                               imidazo[4,5-c]pyridine-6-carboxylic acid                             9        (S)-(-)-4,5,6,7-tetrahydro-1-[(4-methoxy-                                     3-methylphenyl)methyl]-5-(phenylacetyl)-                                      1H-imidazo-[4,5-c]pyridine-6-carboxylic                                       acid                                                                10        (S)-(-)-5-[bis(4-chlorophenyl)acetyl]-                                        4,5,6,7-tetrahydro-1-[(4-methoxy-3-                                           methylphenyl)methyl]-1H-imidazo-                                              [4,5-c]pyridine-6-carboxylic acid                                   11        (S)-(-)-[[4-(acetylamino)-3-                                                  methylphenyl]methyl]-5-(diphenylacetyl)-                                      4,5,6,7-tetrahydro-1H-imidazo-                                                [4,5-c]pyridine-6-carboxylic acid                                   12        (S)-(-)-5-(diphenylacetyl)-1-[(4-hydroxy-                                     3-iodo-5-methylphenyl)meth-                                                   yl]-4,5,6,7-tetrahydro-1H-                                                    imidazo[4,5-c]pyridine-6-                                                     carboxylic acid                                                     13        (S-)-(-)-5-(diphenylacetyl)-1-[(4-hydroxy-                                    3-methylphenyl)methyl]-4,5,6,7-tetrahydro-                                    1H-imidazo[4,5-c]pyridine-6-carboxylic                                        acid                                                                14        (S)-(-)-1-[(4-aminophenyl)-methyl]-5-                                         (diphenylacetyl)-4,5,6,7-tetrahydro-1H-                                       imidazo-[4,5-c]pyridine-6-carboxylic acid                           15        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-[[4-(trifluoromethyl)-                                           phenyl]methyl]-1H-imidazo[4,5-c]pyridine-                                     6-carboxylic acid                                                   16        (S)-(-)-5-(diphenylacetyl)-4,5,6,7-                                           tetrahydro-1-[(3-methylphenyl)methyl]-1H-                                     imidazo[4,5-c]pyridine-6-carboxylic acid                            ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Effect of Example 1 on Rat Striatal                                           Acetylcholine (ACh) Concentrations                                                         Dose                                                             Treatment    (mg/kg, IP) ACh (nM/g ± SEM)                                  ______________________________________                                        Vehicle (Control)                                                                          --          61.0 ± 2.33                                       Example 1     1          57.9 ± 1.86                                       "            10          48.1 ± 0.99*                                      "            30          37.6 ± 2.10*                                      "            60          41.1 ± 1.89*                                      Haloperidol   2          36.9 ± 3.27*                                      DUP 753      30          67.5 ± 5.71                                       ______________________________________                                         Rats were administered test compounds 0.5 hours before animals were           sacrificed. ACh concentrations were determined as described below. Each       value is a mean of at least 4 animals. Results were analyzed by a oneway      analysis of variance followed with NewmanKeuls multiple comparison test.      *Level of significance is p<0.05.                                        

                  TABLE V                                                         ______________________________________                                        Effect of Example 1 on Concentrations of                                      ACh in Rat Striatum and Hippocampus                                                       ACh (nM/g ± SEM)                                               Treatment     Striatum     Hippocampus                                        ______________________________________                                        Vehicle       52.9 ± 1.72                                                                             23.9 ± 2.39                                     (Control)                                                                     Example 1     39.7 ± 0.99*                                                                            15.1 ± 1.55*                                    ______________________________________                                         Animals were treated IP with 30 mg/kg of Example 1. At 0.5 hours post         dosing ACh concentrations were determined as described below.                 *Statistical treatment of data is as described in Table IV.              

                  TABLE VI                                                        ______________________________________                                        Time Course of Effect of Example 1                                            (10 mg/kg, IP) on ACh Concentration in                                        Rat Striatum                                                                  Treatment  Time (h)     ACh (nM/g ± SEM)                                   ______________________________________                                        Control    --           65.4 ± 2.47                                        Example 1  0.5          37.6 ± 2.10*                                       "          1.0          47.9 ± 2.24*                                       "          3.0          50.2 ± 4.53*                                       "          6.0          55.8 ± 4.28                                        "          24.0         61.3 ± 2.30                                        ______________________________________                                         Rats were administered Example 1 and sacrificed at the indicated times.       ACh concentrations were determined as described below.                        *Statistical treatment of data is as described in Table IV.              

                  TABLE VII                                                       ______________________________________                                        Effect of Example 1 on Renal Free Water                                       Clearance                                                                                    Free Water Clearance                                                                             Change                                      Time                              from                                        (min)                mL/min       Predose                                     ______________________________________                                                Vehicle                                                                       Control                                                                30     (predose)    -0.19 ± 0.06                                                                            --                                           60     --           -0.22 ± 0.06                                                                            -16%                                         90     --           -0.26 ± 0.05                                                                            -37%                                        120     --           -0.29 ± 0.05                                                                            -53%                                        150     (postdose)   -0.27 ± 0.06                                          ______________________________________                                                Example 1                                                                     Dose                                                                          (μg/kg/min)                                                         30     0 (predose)  -0.24 ± 0.05                                                                            --                                           60      3           -0.11 ± 0.13                                                                             +54%                                        90      30          +0.11 ± 0.19*                                                                           +150%                                       120     300          +0.64 ± 0.30**                                                                          +370%                                       150     0 (postdose) +0.29 ± 0.10***                                       ______________________________________                                         Data are group means ± standard error. Significant difference from         vehicle control group.                                                        *p = 0.097                                                                    **p = 0.015                                                                   ***p = 0.002                                                             

                  TABLE VIII                                                      ______________________________________                                        Activity of Angiotensin II Antagonists                                        (10 μM) by Brain Region                                                                  .sup.125 I-Angiotensin II Bound                                               (cpm)                                                           Tissue (Rat)                                                                              Total   +SAR    +Example 1                                                                             +DUP 753                                 ______________________________________                                        Cortex       766    336      507     554                                      Cerebellum   571    269      446     346                                      Striatal Membrane                                                                         2462    1295    1838     2067                                     Brain Stem  2124    288     1258     787                                      Cortical Neurons                                                                          2647    293     2118     384                                      ______________________________________                                    

                  TABLE IX                                                        ______________________________________                                        .sup.125 I-Angiotensin II Binding: Inhibition                                 by Subtype Specific Receptor Antagonists                                                  % Inhibition with 1 μM Antagonist                                                        PD 123319                                                         DUP 753     (Example 1)                                         Tissue        (AT.sub.1 Blocker)                                                                        (AT.sub.2 Blocker)                                  ______________________________________                                        Granulosa cell*                                                                              3%         97%                                                 Corpus luteum**                                                                             97%          3%                                                 ______________________________________                                         *Adapted from Pucell, et al, Endocrinology, 1991, 128, 1947-59.               **Data from WarnerLambert Co., Ann Arbor, MI.                            

                  TABLE X                                                         ______________________________________                                        Anatomical Distribution of .sup.125 CI-Example 12                             by Whole Body Autoradiography in Rats                                         Location        Relative Radioactivity                                        ______________________________________                                        Background      0                                                             Testicle        1-2                                                           Uterus          2-3                                                           Thymus          3                                                             Adrenal         3                                                             Heart           3                                                             Blood           4                                                             Lung            4                                                             Bladder*        4-6                                                           Kidney          4-8                                                           Salivary gland  6-7                                                           Ovary (non-follicular)                                                                        3-5                                                           Ovarian follicle                                                                              9                                                             Liver*          10                                                            Intestines*     10                                                            ______________________________________                                         *Represents excretion processes since receptor binding assays show no         AT.sub.2 receptor in these tissues.                                      

                  TABLE XI                                                        ______________________________________                                        Effects of Example 1 on Cyclicity in                                          Female Rats                                                                                   Pre Rx Rx     Post Rx                                         ______________________________________                                        Vehicle                                                                       Length of cycles (days)                                                                          4         4.5  4                                           Number of cycles observed                                                                       10       3      5                                           Example 1                                                                     Length of cycles (days)                                                                          4        14.4    3.9                                       Number of cycles observed                                                                       10       1      4                                           ______________________________________                                         ##STR3##

CHART I ##STR4## wherein (1) --is a single or a double bond;

(2) one of R₁ is present and is

(a) alkyl of from four to twenty carbons, inclusive, ##STR5## wherein yis zero, one, two, three, four, or five, R' is cycloalkyl of from fourto twenty carbons, inclusive in a one-, two-, or three-saturated ringsystem, said ring consisting of from four to eight carbons inclusive,each ring unsubstituted or substituted by a straight or branched loweralkyl group, naphthyl, heteroaryl consisting of 2-, 3-, or 4-pyridyl;1-, 2-, or 4-imidazolyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-indolyl; 2-, or3-thienyl; 2-, or 3-furyl; or 1-, 2-, or 3-pyrazolyl, phenylunsubstituted or substituted with of from one through five substituentsselected from the group consisting of lower alkyl, halo,trifluoromethyl, hydroxy, lower alkoxy, lower alkyl acyloxy, amino,N-lower monoalkylamino, N,N-lower dialkylamino, lower thioalkyl, loweralkylsulfonyl, nitro and ##STR6## wherein R¹⁰ is lower alkyl, phenylunsubstituted or substituted by lower alkyl, or --NHR₁₁ wherein R₁₁ ishydrogen or lower alkyl, and R" is hydrogen, lower alkyl, cycloalkyl offrom four to twenty carbons, inclusive in a one-, two-, orthree-saturated ring system, said ring consisting of from four to eightcarbons inclusive, each ring unsubstituted or substituted by a straightor branched lower alkyl group, naphthyl, phenyl unsubstituted orsubstituted with of from one through five substituents selected from thegroup consisting of alkyl, halo, trifluoromethyl, amino, N-lowermonoalkylamino, N, N-lower dialkylamino, lower thioalkyl, loweralkylsulfonyl, and nitro;

(3) R₂ is

(a) hydrogen,

(b) halo,

(c) lower alkyl,

(d) R'--(CH₂)_(x) wherein x is one, two, three, four, or five and R' isindependently as defined above, ##STR7## wherein R' is independently asdefined above, or (f) R'--CH(OH)-- wherein R' is independently asdefined a3above;

(4) R₃ is

(a) R'--(--CH--)_(x) wherein x and R' are independently as definedabove, ##STR8## wherein R' and y are independently as defined above, andR"' is lower alkyl, cycloalkyl of from four to twenty carbons, inclusivein a one-, two-, or three-saturated ring system, said ring consisting offrom four to eight carbons inclusive, each ring unsubstituted orsubstituted by a straight or branched lower alkyl group, naphthyl,phenyl unsubstituted or substituted with of from one to fivesubstituents selected from the group consisting of alkyl, halo,trifluoromethyl, amino, N-lower monoalkylamino, N,N-lower dialkylamino,lower thioalkyl, lower alkylsulfonyl, and nitro; ##STR9## wherein R₅ is(i) alkyl of from one to fifteen carbons, inclusive, ##STR10## whereinR', R", and y are independently as defined above, (iv) --(--CH═CR₆--)--R₁ wherein R₆ is hydrogen or lower alkyl and R₁ is as definedabove, ##STR11## wherein y, R', and R₆ are independently as definedabove, (vi) R'--(--CH₂ --)_(y) --O-- wherein y and R' are independentlyas defined above, ##STR12## wherein R', R", and y are independently asdefined above, ##STR13## wherein R₅ is independently as defined above;(5) R₄ is

(a) --CH₂ OR₇ wherein R₇ is hydrogen, lower acyl, a lower alkyl,##STR14## wherein R₇ is independently as defined above and R₈ ishydrogen, lower alkyl, or benzyl, ##STR15## wherein R₉ is hydrogen,lower alkyl, or benzyl; and (6) n is one; with the overall proviso thatR₉ cannot be hydrogen, methyl, or ethyl when R₃ is R'--(CH₂)_(x) or##STR16## wherein each of R', R", --(CH₂)_(y) O-- or ##STR17## whereineach of R', R", x, and y are as defined above.

We claim:
 1. A method for regulating the reproductive functionsassociated with AT₂ receptors in a mammal in need thereof whichcomprises administering to said mammal an effective amount of a compoundof Formula I ##STR18## wherein (1) is a single or a double bond;(2) oneof R₁ is present and is(a) alkyl of from four to twenty carbons,inclusive, ##STR19## wherein y is zero, one, two, three, four, or five,R' is cycloalkyl of from four to twenty carbons, inclusive in a one-,two-, or three saturated ring system, said ring consisting of from fourto eight carbons inclusive, each ring unsubstituted or substituted by astraight or branched lower alkyl group, naphthyl, heteroaryl consistingof 2-, 3-, or 4-pyridyl; 1-, 2-, or 4-imidazolyl; 1-, 2-, 3-, 4-, 5-,6-, or 7-indolyl, 2-, or 3-thienyl; 2-, or 3-furyl; or 1-, 2-, or3-pyrazolyl, phenyl unsubstituted or substituted with from one to fivesubstituents selected from the group consisting of lower alkyl, halo,trifluoromethyl, hydroxy, lower alkoxy, lower alkyl acyloxy, amino,N-lower monoalkylamino, N,N-lower dialkylamino, lower thioalkyl, loweralkylsulfonyl, nitro and ##STR20## wherein R¹⁰ is lower alkyl, phenylunsubstituted or substituted by lower alkyl, or --NHR₁₁ wherein R₁₁ ishydrogen or lower alkyl, and R" is hydrogen, lower alkyl, cycloalkyl offrom four to twenty carbons, inclusive in a one-, two-, orthree-saturated ring system, said ring consisting of from four to eightcarbons inclusive, each ring unsubstituted or substituted by a straightor branched lower alkyl group, naphthyl, phenyl unsubstituted orsubstituted with from one to five substituents selected from the groupconsisting of alkyl, halo, trifluoromethyl, amino, N-lowermonoalkylamino, N,N-lower dialkylamino, lower thioalkyl, loweralkylsulfonyl, and nitro; (3) R₂ is(a) hydrogen, (b) halo, (c) loweralkyl, (d) R'--(CH₂)_(x) wherein x is one, two, three, four, or five andR' is independently as defined above, ##STR21## wherein R' isindependently as defined above, or (f) R'--CH(OH)-- wherein R' isindependently as defined above; (4) R₃ is(a) R'--(CH--)_(x) wherein xand R' are independently as defined above, ##STR22## wherein R' and yare independently as defined above, and R"' is lower alkyl, cycloalkylof from four to twenty carbons, inclusive in a one-, two-, orthree-saturated ring system, said ring consisting of from four to eightcarbons inclusive, each ring unsubstituted or substituted by a straightor branched lower alkyl group, naphthyl, phenyl unsubstituted orsubstituted with from one to five substituents selected from the groupconsisting of alkyl, halo, trifluoromethyl, amino, N-lowermonoalkylamino, N,N-lower dialkylamino, lower thioalkyl, loweralkylsulfonyl, and nitro; ##STR23## wherein R₅ is (i) alkyl of from oneto fifteen carbons, inclusive, ##STR24## wherein R', R", and y areindependently as defined above, (iii) --(--CH═CR₆ --)--R₁ wherein R₆ ishydrogen or lower alkyl and R₁ is as defined above, ##STR25## wherein y,R', and R₆ are independently as defined above, (v) R'--(--CH₂ --)_(y)--O-- wherein y and R' are independently as defined above, ##STR26##wherein R', R", and y are independently as defined above, ##STR27##wherein R₅ is independently as defined above; (5) R₄ is (a) --(CH₂ OR₇wherein R₇ is hydrogen, lower acyl, a lower alkyl, ##STR28## wherein R₇is independently as defined above and R₈ is hydrogen, lower alkyl, orbenzyl, ##STR29## wherein R₉ is hydrogen, lower alkyl, or benzyl; and(6) n is one; with the overall proviso that R₉ cannot be hydrogen,methyl, or ethyl when R₃ is R'--(CH₂)_(x) or ##STR30## wherein R₅ isR'--(CH₂)_(y) O-- or ##STR31## wherein each of R', R", x, and y are asdefined above or a pharmaceutically acceptable salt thereof.
 2. Themethod of claim 1 wherein the compoundis:(S)-(-)-1-[[4-dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c ]-pyridine-6-carboxylic acid, (S)-(-) -1-[(4-amino-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid,(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-[(4-methoxy-3-methyl-phenyl)methyl]-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-(2-tricyclo[3,3,1,1³,7]dec-1-ylethyl-1H-imidazo[4,5-c]pyridine-6-carboxylic acid,(S)-(-)-5-[bis(4-fluorophenyl)acetyl]-1-[[4-(dimethylamino)-3-methyl-phenyl]methyl]-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(9H)-fluoren-9-ylcarbonyl)-4,5,6,7-tetrahydro-1-[(4-methoxy-3-methylphenyl)methyl]-1H-imidazo-[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-[(3-methyl-4-nitrophenyl)-methyl]-4-nitrophenyl)methyl]-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-(phenylmethyl)-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-4,5,6,7-tetrahydro-1-[(4-methoxy-3-methylphenyl)methyl]-5-(phenylacetyl)-1H-imidazo-[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-[bis(4-chlorophenyl)acetyl]-4,5,6,7-tetrahydro-1-[(4-methoxy-3-methylphenyl)methyl]-1H-imidazo-[4,5-c]pyridine-6-carboxylic acid,(S)-(-)-[[4-(acetylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo-[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-1-[(4-hydroxy-3-iodo-5-methylphenyl)methyl]-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-1-[(4-hydroxy-3-methylphenyl)methyl]-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-1-[(4-aminophenyl)-methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo-[4,5-c]pyridine-6-carboxylicacid,(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-[[4-(trifluoromethyl)phenyl]methyl]-1H-imidazo[4,5-c]pyridine-6-carboxylicacid, or(S)-(-)-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1-[(3-methylphenyl)methyl]-1H-imidazo[4,5-c]pyridine-6-carboxylic acid.
 3. The method of claim 2 wherein thefunction to be regulated is selected from the menstrual cycle,fertility, and hormonal balances of the estrus cycle.